Abstract Objective We developed an assay to measure the concentration of 25 hydroxyvitamin D₂ and D₃ in protein extracts derived from stored neonatal dried blood spots. During this study, we postulated that these samples had been contaminated with exogenous vitamin D metabolites because of the addition of bovine serum albumin (BSA) as part of an extraction step undertaken 7 years earlier. The aim of the current study was to develop methods in order to adjust for this contamination. Results We identified between-plate variations in 25 hydroxyvitamin D₂ and D₃ concentrations which suggested the presence of three different BSA batches. Based on repeat extraction (without the addition of BSA) and testing of 395 samples, we developed models to correct for the exogenous 25 hydroxyvitamin D₂ and D_3. The regression models were Diff_25OHD3 = − 8.2 + 1.8* Diff_25OHD2 for low contamination, Diff_25OHD3 = 23.8 + 1.7* Diff_25OHD2 for middle contamination, and Diff_25OHD3 = 14.3 + 3.0* Diff_25OHD2 for high contamination. After these corrections, the three subsamples had comparable distributions within the expected range for both 25 hydroxyvitamin D₂ and D₃.